1. Field of the Invention
This invention concerns an expression vector system comprising a pair of expression vectors constructed from a wild-type and a mutant version of any marker gene, including a reporter or selection gene, and a method for optimization and confirmation of DNA delivery and of gene targeting and for quantification of targeting frequency. In particular, the invention concerns and embodies pairs of novel prokaryotic/eukaryotic DNA expression vectors used for DNA delivery and gene targeting assessment and targeting frequency quantification. The vector pairs permit assessment and confirmation of DNA delivery as well as optimization of the DNA delivery efficiency and gene targeting in eukaryotic systems and quantification of targeting frequency. The invention further permits assessment of nuclear delivery of the DNA, assessment of cell enzymatic function and assessment of cell enzymatic pathways.
2. Background and Related Disclosures
Gene targeting, as a strategy for gene therapy, provides a mechanism for achieving permanent, site-specific correction of DNA lesions and results in the cell-appropriate expression of the target gene (Gene Ther., 5:149 (1998) and Gene Ther., 6:1347 (1999)).
A gene targeting strategy allows the integrity and regulation of the gene to be maintained even when cis-acting regulatory elements are distant from the coding sequences as described in Trends in Genetics, 15:403 (1999) or are within introns as described in J. Biol. Chem., 271:9947 (1996). In addition, the non-viral gene delivery strategies used to introduce the targeting DNA into cells, mitigate the immune and inflammatory side-effects triggered by viral capsid antigens and unmethylated CpG sequences present in cDNA expression plasmids as described in PNAS (USA), 91:4407 (1994), PNAS (USA), 83:2879 (1996) and Gene Ther., 6:1448 (1999).
Small fragment homologous replacement (SFHR) is a gene targeting strategy that has been successfully used to target and modify genomic DNA sequences through the introduction of small fragments of DNA homologous to specific genomic loci in human epithelial cells. SFHR was disclosed in the U.S. Pat. No. 6,010,908, issued on Jan. 4, 2000,hereby incorporated by reference. SFHR was initially used to correct the ΔF508 mutation, the most common lesion associated with the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in CF airway epithelial cells. The DNA fragments were comprised of a 491-bp wild-type (wt) CFTR sequence that would replace the three deleted nucleotides that characterize the ΔF508 mutation. As a result, wtCFTR MRNA expression and the functional correction of the cAMP-dependent Cl-transport defect associated with CF was observed and is further described in Gene Ther., 3:859 (1996). A targeting frequency of 1-10% was indicated in this study.
In a parallel study, targeting DNA fragments comprising genomic ΔF508 CFTR sequence (488-bp) were able to delete the three nucleotides that constitute the ΔF508 mutation gene in the genomic DNA of normal human airway epithelial cells as described in Hum. Mol. Genet., 7:1913 (1998).
For the successful application of SFHR as a gene therapy, optimization of the conditions for targeting is necessary. A drawback of having CFTR as the target gene is the lack of an endogenous selection mechanism that readily differentiates between targeted and parental cells and, thus, limits the ability to accurately quantify targeting frequency.
Additionally, there are currently no established biological assays available for rapid determination of the amount of transfected DNA that was successfully delivered into the cell nucleus and there are no commercial products, methods or assays for determination of site-specific modification of genomic sequences.
While the SFHR method provides a convenient and effective way to alter DNA sequences, determination of the replacement efficiency is still problematic. Inaccessible gene targeting elements and/or target vector and/or a cell system that does not support gene targeting can decrease the gene targeting efficiency. Low gene targeting frequencies can be caused by a DNA delivery system that interferes with the enzymatic pathway(s)/machinery that mediates modification of the target sequences.
It would, therefore, be advantageous to have available a system and/or method and/or means to permit a confirmation of the nuclear DNA delivery efficacy, the optimization of such delivery and quantification of targeting frequency.
It is, therefore, a primary objective of the current invention to provide a means and method for optimization of the DNA delivery efficiency, confirmation of the DNA delivery and quantification of targeting frequency of SFHR-mediated modification of target DNA.
All patents, patent applications and publications described herein are being incorporated by reference.